Arthrospira platensis nanoparticles dietary supplementation improves growth performance, steroid hormone balance, and reproductive productivity of Nile tilapia (Oreochromis niloticus) broodstock

This study evaluates the impact of dietary supplementation of the blue-green alga Arthrospira platensis NIOF17/003 nanoparticles (AN) on the growth performance, whole-body biochemical compositions, blood biochemistry, steroid hormonal, and fry production efficiency of Nile tilapia (Oreochromis niloticus) broodstock, during the spawning season. After a 21-day preparation period to equip the females and ensure that their ovaries were filled with eggs, mating between the mature females and males took place in a 3:1 ratio during a 14-day spawning cycle. A total of 384 tilapia broodstock 288 females and 96 males with an initial body weight of 450.53±0.75, were divided into four groups; AN0: a basal diet as a control group with no supplementation of Arthrospira platensis, and the other three groups (AN2, AN4, and AN6) were diets supplemented with nanoparticles of A. platensis at levels of 2, 4, and 6 g kg─1 diet, respectively. The results found that fish-fed group AN6 showed the highest significant differences in weight gain (WG), final weight (FW), feed conversion ratio (FCR), protein efficiency ratio (PER), and feed efficiency ratio (FER). Females fed the AN6 diet showed the highest significant fat content. Compared to the AN0 group, fish fed on the supplemented diets showed significant improvement (p < 0.05) in triglyceride, glucose, and aspartate aminotransferase (AST). A gradual increase in AN inclusion level resulted in a gradual increase in the concentrations of luteinizing hormone (LH), and follicle-stimulating hormone (FSH), testosterone, progesterone, and prolactin. The rates (%) of increase in fry production for females fed supplemented diets were 10.5, 18.6, and 32.2% for AN2, AN4, and AN6, respectively, compared to the control group. This work concluded that the inclusion levels of 6 g kg─1 of A. platensis nanoparticles in the diet of Nile tilapia broodstock significantly improved the growth performances, steroid hormone concentrations, and increased the fry production efficiency by 32.2%, respectively. These findings revealed that A. platensis nanoparticles resulted in a significantly enhanced female’ reproductive productivity of Nile tilapia broodstock.


Introduction
Aquaculture development and sustainability are directly influenced by several factors as global environmental, economic, and political issues such as feed ingredient availability, diet cost production, wars, pandemics, water quality, climate change, ocean productivity, plankton communities, productivity, and nutritional values [1,2].Globally, tilapia culture has experienced a sharp expansion over the past two decades and is farmed in more than 130 countries worldwide [3].Tilapia is currently the second most important farmed finfish species in the world [4].Global production of farmed tilapia grew by 3.3% in 2020 to top 6 million tons for the first time, despite the impact of COVID-19.The expansion of tilapia production all over the world is due to its ability to be produced in various aquatic environments, selective breeding, and its potential to replace marine fish products [1,5,6].
Although several factors limit aquaculture development, such as feeding costs, diseases, bad water quality, the low performance of broodstock, and the high mortality rate in seeds [7,8], several strategic approaches have been recently adopted in aquaculture to sustain tilapia production [9].One such approach is the production of functional feed that contains health promoters and immune stimulants.Functional feed additives have become the main component of any strategy to control disease outbreaks in aquaculture, particularly when opportunistic bacteria are suspected to be a major cause of mortality [10].Several functional feed additives have been utilized such as binders, algae derivatives, antimicrobials, seaweed extracts, antioxidants, and enzymes, which improve feed and water quality [11][12][13][14][15][16].Other feed additives improve animal performance and health such as immunostimulants probiotics, photogenic, and prebiotics [17][18][19][20][21].
Besides its applications as aqua feed additives, Arthrospira or its derivatives have many biotechnological applications.The lipid-free dry weight (biodiesel byproduct) of A. platensis NIOF17/003 was successively utilized as dry feed for rotifer (Brachiounus plicatilis), in the same line to remove ammonia and organic dye from aquaculture wastewater effluents and industrial textile effluents, respectively [63,64].The growth of aquaculture has raised the demand for better diets and aqua-feed additives.Recently, several studies have documented different forms of aqua-feed additives in aquaculture feed.The form of feed-additive inclusion is of high importance to maximize the utilization of added materials [65].Recently, interest in using nanoparticles of several materials as animal feed additives has been expanded attributed to the higher bioavailability and efficiency [57].
Nanotechnology applications have been successfully increased [66][67][68].Algae nanoparticle applications in aqua feed diets also increased due to their high of their high surface area of nanoparticles [69] which enhances growth performance, feed utilization, body composition, stress tolerance, and enhanced immune system for many species such as Nile tilapia [42,70], Pacific white shrimp [42], black tiger shrimp [57], and Zebrafish [71].The current study aims to evaluate the effect of the cyanobacterium species, Arthrospira platensis NIOF17/003 nanoparticles, as a functional feed additive on growth performances, whole-body biochemical composition, blood biochemistry, steroid hormonal status, and seeds production efficiency for the Nile tilapia broodstock during the spawning cycle.

Water quality indices
Following the guidelines of APHA [73], water quality parameters of ammonia (NH 3 , mg L ─1 ), nitrate (NO 3 , mg L ─1 ), nitrite (NO 2 , mg L ─1 ), and dissolved oxygen (DO, mg L ─1 ) were determined three times a week.Furthermore, pH, salinity (ppt), and temperature (˚C) were determined daily (1.00 pm) during the experimental period and Table 1 shows the water quality parameter during the experimental period.During the 14-days spawning cycle and the 21-day equipping period, all remarkable water qualities were within the recommended range of production requirements of Nile tilapia broodstock during the spawning season.

Nile tilapia (Oreochromis niloticus) broodstock
2.3.1.Experimental fish and design.The current experiment was carried out in a private Tilapia hatchery located in Port Said Governorate, Egypt.Nile tilapia broodstock (males and females) was obtained from a commercial farm of Nile tilapia located in Port Said Governorate, Egypt.The fish were given a control-based diet for 21 days before starting the feeding trial to initiate the spawning cycle.After 21 days of acclimation, during which the females' ovaries were investigated to be ready for spawning, mating took place between males and females in a 3:1 ratio for 14 days.In the current experiment, the main source of water was the irrigation water of the El-Slam Canal, and the rate of daily freshwater change was 30%.Daily, fish faeces, unconsumed feed, and wastes were removed by siphoning.The tanks were aerated using an air blower.The experiment was conducted with four groups, in a greenhouse with 12 concrete tanks of 8 m 3 (2 m x 4 m x 1 m) each.A total 384 of Nile tilapia broodstock (288 females and 96 males) were divided into four groups, each having three replicates.

Experimental diet.
As presented in Table 2, four diets were used in this study: AN 0 : a basal diet as a control group, while the other three groups (AN 2 , AN 4 , and AN 6 ) were basal diet supplemented with nanoparticles of A. platensis at levels of 2, 4, and 6 g kg ─1 diet, respectively.The addition of respective levels of A. platensis nanoparticles to diets was performed as previously described by Mabrouk et al. [8].Briefly, The prepared nanoparticle powder of A. platensis nanoparticle was dissolved in an adequate volume of distilled water and set aside to be mixed with the remaining diet ingredients.The diet ingredients were formulated by thoroughly combining and the A. platensis nanoparticles were sprayed on four sets of the experimental diets at the rates of 0, 2, 4, and 6 g/kg diet.Following this, the oil and water were mixed extensively with the ingredients, and the mixture was pelleted using the Sprout-Waldron Laboratory Pellet Mill (CPM, California Pellet Mill Co., USA) to create 2 mm pellets.The pellets were then dried in ovens at 40˚C until the moisture level dropped below 10% [8].The biochemical composition of basal diet, based on the % of dry matter bases, of crude protein (29.9%), ether extract (9.2%), crude fiber (4.7%), nitrogen-free extract (48.7%), ash (7.5%), gross energy (4963 kj kg ─1 diet), and digestible energy (3520 kj kg ─1 diet) were calculated according to the reported guideline of AOAC [74].Fish were hand-fed three times daily, at 9 am, 12 pm, and 4 pm, at a rate equivalent to 3% of their wet body weight.

Tested parameters 2.4.1. Growth indices.
At the end of the rearing trial, fish were starved for 24 h to empty the digestive tract [75].After that, the total body weight and the total number of each replicate were investigated to calculate weight gain (WG), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency ratio (PER), and survival rate (SR) using the given formulas.
To calculate weight gain (WG), the initial weight (IW, 450.53 ± 0.75) and final weight (FW) of mothers were determined before and after the 14-days spawning cycle experiment.No mortality was observed during the experiment.Moreover, the indices of feed conversion ratio (FCR), protein intake (PI), protein efficiency ratio (PER), and feed efficiency ratio (FER) were calculated as described below.

WG ðgÞ ¼ FBW À IBW
where; FBW and IBW are the final and initial body weight ðgÞ; respectively: ð1Þ 2.4.2.Biochemical analysis.At the end of a 14-day spawning cycle experiment, fish in all dietary treatments were starved for 24 h, and five fish (three females and two males) were randomly collected, homogenized, dried, ground, and stored under -20˚C for whole-body analysis as described elsewhere [41].The whole-body biochemical composition of fish and diets were determined, and moisture, dry matter (DM), crude protein (CP), ether extract (EE), crude fiber (CF), nitrogen-free extract (NFE), gross energy (GE), and digestible energy (DE) were determined and calculated according to the guideline of AOAC [74].
2.4.3.Blood serum analysis.At the end of the experiment, six fish samples (three males and three females) from each replicate were anesthetized using TMS buffered (Tricaine Methanesulfonate at the dose of 30 mg L ─1 ) to collect blood serum for blood biochemistry analysis protocol as previously described by Ferguson et al. [76].Blood samples were extracted using a sterilized hypodermic syringe (3 mL with a 22-gauge needle and a heparinized tube) and stored at room temperature for 30 minutes and then centrifuged at 3.000 RPM for 15 m.The collected serum was stored at -20˚C for further analysis.The concentration of total protein (g dL ─1 ) [77] and albumin (g dL ─1 ) [78] were measured, and the globulin level (g dL ─1 ) was calculated as the difference between the values of total protein and albumin.The levels of glucose (mg dL ─1 ) [79] and triglyceride (TAG) (mg dL ─1 ) [80] levels were measured using kits from El-Nasr Pharmaceutical Chemicals Co., Egypt, following the provided instructions.Moreover, using the calorimetric techniques, the activities of serum glutamic pyruvate transaminase (GPT, U mL ─1 ) [81].Aspartate aminotransferase (AST, U mL ─1 ), and alanine (ALT, U mL ─1 ) were determined according to [82] using commercial kits from Biodiagnostic in Egypt, as per the manufacturer's guidelines.

Steroid hormones (SHs).
At the end of the spawning trial, from each replicate, six mothers' samples (three males and three females) were randomly selected to determine SHs.For both males and females, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were determined.For males only, total testosterone (TT) and free testosterone (FT) were determined.For females only, prolactin and progesterone hormones were determined.The SHs were calorimetry determined using ELISA assay as described elsewhere [83], an enzyme-linked immune sorbent, known as the Immulite/Immulite 1000 system [84].The fishspecific commercial kits of FSH (RAB0660-1KT), LH (SE120071), TT (SE120120), FT (SE120120), PRO (RAB0408-1KT), and PRG (SE120087) were determined, following to the manufacturer's instructions.
2.4.5.Females reproductive productivity.After the 14-days spawning trial, the adult fish were carefully gathered and transferred to different ponds, while lowering the water level.The method described by El-Sayed et al. [85] was followed to collect the eggs.The number of offspring produced by each female was calculated using the following formula:

Number of fries per female ¼ Total number of seeds=pond Total number of females=pond ð6Þ
The average ratio (%) of the number of seeds from mothers fed the control diet to the supplemented diets was conducted as the following Eq.: Where Sn and Sc are the numbers of seeds that come from mothers fed the supplemented and the control diets, respectively.

Statistical analysis
The data were assessed for homoscedasticity and normality before conducting statistical analysis.The results were presented as mean ± standard deviation (n = 3).The statistical analysis was performed using the SPSS computer software package.To determine significant differences among means at a p-value of less than 0.05, a one-way ANOVA was performed followed by Duncan's multiple-range tests.The graphical representation of the steroid hormone levels and broodstock seed production figures was created using GraphPad Prism version 9.

Growth indices
The growth performances of Nile tilapia, during the spawning cycle, are presented in Table 3. Significant differences (p < 0.05) were revealed in FW, WG, FCR, PER, and FER between the control group (AN 0 ) and the groups supplemented with A. platensis nanoparticles (AN 2 , AN 4 , and AN 6 ).However, fish-fed group AN 6 showed the highest significant differences (p < 0.05) in FW, WG, FCR, PER, and FER.

Blood biochemistry
Table 4 shows the blood serum biochemical analysis of Nile tilapia fed different inclusion levels of A. platensis nanoparticles.Among the dietary groups, no significant differences (p < 0.05) were observed in the total protein, albumin, globulin, GPT, and ALT contents.Compared to the control group, fish feed A. platensis nanoparticles (AN 2 , AN 4 , and AN 6 ) showed significant differences (p < 0.05) in TAG, glucose, and AST.The highest significant (p < 0.05) TAG was observed in fish fed the AN 4 diet, followed by AN 6 , and AN 2 , while the lowest TAG content was observed in control fish.The highest significant (p < 0.05) glucose was observed in fish fed with AN 6 , followed by AN 4 , and AN 2 , while the lowest glucose was revealed in fish fed the control diet.On the other hand, compared to the A. platensis nanoparticles groups, controlfed fish showed significantly (p < 0.05) the highest AST content, followed by AN 2, AN 4 , and AN 6 .

Discussion
According to the literature, algal cells (microalgae or seaweeds) and bioactive compounds extracted from algal such as astaxanthin from A. platensis [41], and polysaccharides extracted from brown seaweed (Sargassum dentifolium) [8,42,86] were included as feed additives in various forms as dry powder, liquid extract [65].The nanoparticle form is the newest technology that can improve diet efficiency [70].A study conducted by Nagarajan et al. [87] reported that these positive effects of nanoparticle forms may be due to the novel properties of highly fine particles and the high surface area of the molecules.These new properties can change, maximize, and create novel properties for the phytochemical compounds in the microalgae nanoparticle forms compared to the traditional microalgae form [88][89][90][91].
To the best of our knowledge, no study has investigated the effects of nanoparticles of microalga A. platensis on Nile tilapia during the spawning period.The present study showed improvement in growth performances, whole-body biochemical composition, physiological aspects, steroid hormonal status, and fry production efficiency for the Nile tilapia during the spawning cycle.The current study revealed that the AN 6 group has achieved the highest significant differences (p < 0.05) in FW, WG, FCR, PER, and FER, compared to the control group (AN 0 ) and the other supplemented groups (AN 2 and AN 4 ).Elabd et al. [70] revealed that inclusion levels of 2.5-5 g kg ─1 of A. platensis nanoparticles into Nile tilapia diets significantly improved growth performance indices.In a later study, Sharawy et al. [42] reported that the inclusion of A. platensis nanoparticles (2.5, 5, and 10 g kg ─1 diet) in a Pacific white shrimp diet significantly (p < 0.05) improved the growth performances of shrimp fry.The findings reported in the present study reported that the inclusion concentrations (6 g kg ─1 diet) achieved an economic advantages regarding the growth performance.However, this small increase in FW and WF may be attributed to the fact that the broodstock at this age tends to direct all its energy to the steroid hormonal aspects, ovulation, and egg production, not to the growth and building tissues.
Aquafeeds are significantly affecting the carcass composition of aquatic animals, especially in early growth stages [93,94].In the current study, supplemented groups significantly affected the protein and the lipid content of Nile tilapia mothers.These results are in accordance with the previous studies which concluded that the nanoparticles form of A. platensis inclusion levels to Nile tilapia significantly improves protein and lipid contents [8,42,70].This finding may be attributed to the fact that A. platensis is a rich source of protein (50-65%) and has a highquality fatty acid profile (AA, EPA, and DHA) with total lipid content of 4-8% [39].
Blood biochemistry indices (serum protein, albumin, TAG, glucose, GPT, AST, and ALT) are major factors in improving blood aspects, immune system, and overall physiological status of fish and act powerfully as adjuncts to assess the efficiency of feed additives [95][96][97].The current study reported that the only significant difference in blood biochemistry indices was recorded in TAG, and glucose by the supplemented diets compared to the control diet.These results are in accordance with previous results of Mabrouk et al. [8], Elabd et al. [70], and Sharawy et al. [42].However, these results may be due to the high-lipid content of A. platensis nanoparticles dietary supplementations [98].
To achieve successful and effective reproduction of aquatic animals, it's essential to understand the relationship between hormonal spawning and nutritional and environmental factors such as diet development, photoperiod, and temperature which sequentially, affect seed production efficiency [99,100].In the current study, different nanoparticle inclusion levels of A. platensis significantly (p < 0.05) affect hormonal spawning for Nile tilapia broodstock (males and females).The current study observed that a gradual increase in incorporation nanoparticle levels resulted in a gradual increase in hormonal spawning (FSH, LH, free testosterone, total testosterone, progesterone, and prolactin).Compared to the control group (AN 0 ), increasing rates in fry production for the supplemented diets of AN 2 , AN 4 , and AN 6 were 10.5, 18.6, and 32.2%, respectively were revealed.These findings were in accordance with the results of several studies which concluded that Arthrospira (Spirulina) strains can improve the formation of ovulation, prostaglandin, and steroidogenesis, improve maturation ability, optimize the levels of sex hormones, enhance reproduction performance and hatching efficiency, and increase seeds production in fish species such as Nile tilapia [101], catfish (Clarias gariepinus) [102], zebrafish females [103], three-spot gourami (Trichopodus trichopterus) [104], yellow tail cichlid (Pseudotropheus acei) [105], parrot fish (Oplegnathus fasciatus) [106], Pla Pho (Pangasius bocourti) [107], and goldfish (Carassius auratus) [108].Promya and Chitmanat [102] concluded that Arthrospira is an alternative candidate to artificial hormones in the diet of fish brooders.Interestingly, due to their novel physical properties and bioactive compounds, the nanoparticle form of A. platensis maximizes the nutritional benefit for Nile tilapia.
Joshua and Zulperi [98] reported that the nutritional impact and bioactive material contents of A. platensis and Chlorella vulgaris, they are algal species that can significantly enhance the immune system, reduce disease infections, improve the hormonal spawning, and improve reproduction aspects of fish and shrimp.Hassaan [109] concluded that the inclusion of 10-15 g kg ─1 of microalgae Cyclotella spp.(dried form) in the diet of Nile tilapia broodstock significantly improved hormonal spawning, gonadosomatic index, condition factor, semen quality, and relative absolute fecundity, which consequentially improved seed production.

AN 4 ,
and AN 6 ) achieved the highest protein content (Fig1A).The highest significant (p < 0.05) lipid content was observed by fish fed the group of AN 6 , followed by AN 2 , AN 4 , and AN 0 (Fig1B).Among the dietary groups, no significant differences (p < 0.05) were observed in ash content (Fig 1C), and the lowest moisture content was observed by mothers fed the control group (AN 0 ) (Fig1D).

Figs 2 -
Figs 2-4 show the influence of different inclusion levels of A. platensis nanoparticles on steroid hormone concentrations in Nile tilapia males and females.Fig 2 displayed that fish-fed supplemented diets revealed a significant (p < 0.05) improvement in FSH and LH concentrations in both females and males, compared to control-fed fish.Moreover, a gradual increase in incorporation levels of A. platensis nanoparticles resulted in a gradual increase in LH and FSH concentrations.Furthermore, the females showed a positive response to the gradual increase in inclusion levels higher than males (Fig 2).Fig 3 shows that the gradual increase in inclusion levels of A. platensis nanoparticles resulted in a gradual increase in the concentrations of testosterone (total and free).Compared to the control group, significant (p < 0.05) improvements in total and free testosterone concentrations were obtained by males fed diets supplemented

Fig 5
Fig 5  shows the impact of different inclusion levels of A. platensis nanoparticles on the seed's production count.A gradual increase in incorporation levels resulted in a significant (p < 0.05) gradual increase in seed production efficiency.Compared to control-fed fish, increasing rates in seed production were noticed for AN 2 , AN 4 , and AN 6 diets, 10.5, 18.6, and 32.2%, respectively.

Fig 5 .
Fig 5. Impact of different nanoparticles inclusion levels of A. platensis on the seed production efficiency of O. niloticus.AN 0% , AN 2 , AN 4 , and AN 6 are diets supplemented with A. platensis nanoparticles at levels of 0, 2, 4, and 6 g kg ─1 diet, respectively.The presented data are Means ± SD (n = 3).Different letters are significantly different (p < 0.05).The percentages in the bars are the increased percentage (%) in seed production for females fed supplemented diets compared to the control diet.https://doi.org/10.1371/journal.pone.0299480.g005

Table 1 . Water quality parameters during the spawning cycle experiment.
AN 0 , AN 2 , AN 4 , and AN 6 : Diets supplemented with A. platensis nanoparticles at levels of 0, 2, 4, and 6 g kg ─1 diet, respectively.The presented data are Means ± SD (n = 3).Different letters in the same raw are significantly different (p < 0.05).The absence of letters in the same row means no significant differences. *https://doi.org/10.1371/journal.pone.0299480.t001

Table 3 . Growth performance indices of O. niloticus broodstock during the spawning season.
Fig 1shows the approximate whole-body compositions (protein, lipid, ash, and moisture) of Nile tilapia fed the control diets and the diets supplemented with A. platensis nanoparticles.Compared to the control group, both males and females fed with supplemented diets (AN 2 , * AN 0 , AN 2 , AN 4 , and AN 6 : Diets supplemented with A. platensis nanoparticles at levels of 0, 2, 4, and 6 g kg ─1 diet, respectively.The presented data are Means ± SD (n = 3).Different letters in the same raw are significantly different (p < 0.05).The absence of letters in the same row means no significant differences (p < 0.05).WG: Weight gain, FW: Final weight, IW: Initial weight, FCR: Feed conversion ratio, PI: Protein intake, PER: efficiency ratio, and FER: Feed efficiency ratio.https://doi.org/10.1371/journal.pone.0299480.t003